The Experts below are selected from a list of 162 Experts worldwide ranked by ideXlab platform

Taroh Kinoshita - One of the best experts on this subject based on the ideXlab platform.

  • Glycosylphosphatidylinositol mannosyltransferase II is the rate-limiting enzyme in glycosylphosphatidylinositol biosynthesis under limited Dolichol-Phosphate Mannose availability
    Journal of Biochemistry, 2013
    Co-Authors: Tetsuya Hirata, Noriyuki Kanzawa, Yusuke Maeda, Morihisa Fujita, Yoshiko Murakami, Taroh Kinoshita
    Abstract:

    Although the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) are well characterized, the regulation of GPI biosynthesis remains unclear. We isolated and characterized a mutant cell line showing decreased surface expression of CD59 and the accumulation of GPI intermediates. The mutant cell line was partially defective in MPDU1, which encodes a protein required for the utilization of Dolichol-Phosphate Mannose. Overexpression of PIGV, which encodes GPI mannosyltransferase II, restored the surface expression of CD59 and normalized the accumulation of GPI intermediates in the mutant cells. Among all known genes involved in GPI biosynthetic pathway, only PIGV had such suppressive activity. PIGV, however, did not restore the abnormality of N-glycosylation caused by MPDU1 mutation. Our results suggest that GPI mannosyltransferase II is the rate-limiting enzyme in GPI biosynthesis under limited Dolichol-Phosphate Mannose availability.

  • Dolichol-Phosphate Mannose synthase: Structure, function and regulation
    Biochimica et biophysica acta, 2008
    Co-Authors: Yusuke Maeda, Taroh Kinoshita
    Abstract:

    Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and Dolichol-Phosphate-sugars, in which the sugar moiety is Mannose or glucose. The sugar transfer from Dolichol-Phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of Dolichol-Phosphate-Mannose, and particularly on the mammalian enzyme complex involved in the reaction.

  • DPM1, the Catalytic Subunit of Dolichol-Phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3
    The Journal of biological chemistry, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Taroh Kinoshita
    Abstract:

    Abstract Dolichol-Phosphate Mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-Mannose, and C-Mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.

  • mammalian pig x and yeast pbn1p are the essential components of glycosylphosphatidylinositol mannosyltransferase i
    Molecular Biology of the Cell, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Taroh Kinoshita
    Abstract:

    Within the endoplasmic reticulum (ER), Mannoses and glucoses, donated from Dolichol-Phosphate-Mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four Mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using Dolichol-Phosphate-monosaccharide.

  • Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.
    Molecular biology of the cell, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Youn Uck Kim, Taroh Kinoshita
    Abstract:

    Within the endoplasmic reticulum (ER), Mannoses and glucoses, donated from Dolichol-Phosphate-Mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four Mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was

Yusuke Maeda - One of the best experts on this subject based on the ideXlab platform.

  • Glycosylphosphatidylinositol mannosyltransferase II is the rate-limiting enzyme in glycosylphosphatidylinositol biosynthesis under limited Dolichol-Phosphate Mannose availability
    Journal of Biochemistry, 2013
    Co-Authors: Tetsuya Hirata, Noriyuki Kanzawa, Yusuke Maeda, Morihisa Fujita, Yoshiko Murakami, Taroh Kinoshita
    Abstract:

    Although the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI) are well characterized, the regulation of GPI biosynthesis remains unclear. We isolated and characterized a mutant cell line showing decreased surface expression of CD59 and the accumulation of GPI intermediates. The mutant cell line was partially defective in MPDU1, which encodes a protein required for the utilization of Dolichol-Phosphate Mannose. Overexpression of PIGV, which encodes GPI mannosyltransferase II, restored the surface expression of CD59 and normalized the accumulation of GPI intermediates in the mutant cells. Among all known genes involved in GPI biosynthetic pathway, only PIGV had such suppressive activity. PIGV, however, did not restore the abnormality of N-glycosylation caused by MPDU1 mutation. Our results suggest that GPI mannosyltransferase II is the rate-limiting enzyme in GPI biosynthesis under limited Dolichol-Phosphate Mannose availability.

  • Dolichol-Phosphate Mannose synthase: Structure, function and regulation
    Biochimica et biophysica acta, 2008
    Co-Authors: Yusuke Maeda, Taroh Kinoshita
    Abstract:

    Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and Dolichol-Phosphate-sugars, in which the sugar moiety is Mannose or glucose. The sugar transfer from Dolichol-Phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of Dolichol-Phosphate-Mannose, and particularly on the mammalian enzyme complex involved in the reaction.

  • DPM1, the Catalytic Subunit of Dolichol-Phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3
    The Journal of biological chemistry, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Taroh Kinoshita
    Abstract:

    Abstract Dolichol-Phosphate Mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-Mannose, and C-Mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.

  • mammalian pig x and yeast pbn1p are the essential components of glycosylphosphatidylinositol mannosyltransferase i
    Molecular Biology of the Cell, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Taroh Kinoshita
    Abstract:

    Within the endoplasmic reticulum (ER), Mannoses and glucoses, donated from Dolichol-Phosphate-Mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four Mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using Dolichol-Phosphate-monosaccharide.

  • Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.
    Molecular biology of the cell, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Youn Uck Kim, Taroh Kinoshita
    Abstract:

    Within the endoplasmic reticulum (ER), Mannoses and glucoses, donated from Dolichol-Phosphate-Mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four Mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was

Malcolm J Mcconville - One of the best experts on this subject based on the ideXlab platform.

  • regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in leishmania mexicana
    Molecular Biology of the Cell, 2001
    Co-Authors: Kylie A Mullin, Steven C Ilgoutz, Jody L Zawadzki, Bernardo J Foth, Judy M Callaghan, Geoffrey I Mcfadden, Malcolm J Mcconville
    Abstract:

    The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of Dolichol-Phosphate-Mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.

  • glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in leishmania mexicana
    The EMBO Journal, 1999
    Co-Authors: Steven C Ilgoutz, Kylie A Mullin, Bridget R Southwell, Malcolm J Mcconville
    Abstract:

    Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana Dolichol-Phosphate-Mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.

  • evidence that free gpi glycolipids are essential for growth of leishmania mexicana
    The EMBO Journal, 1999
    Co-Authors: Steven C Ilgoutz, Jody L Zawadzki, Julie E Ralton, Malcolm J Mcconville
    Abstract:

    The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for Dolichol-Phosphate-Mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of Dolichol-Phosphate Mannose, the sugar donor for all Mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked Mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for Dolichol-Phosphate-Mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.

Jerry Eichler - One of the best experts on this subject based on the ideXlab platform.

  • agls a novel component of the haloferax volcanii n glycosylation pathway is a Dolichol Phosphate Mannose mannosyltransferase
    Journal of Bacteriology, 2012
    Co-Authors: Chen Cohenrosenzweig, Sophie Yuristdoutsch, Jerry Eichler
    Abstract:

    In Haloferax volcanii, a series of Agl proteins mediates protein N-glycosylation. The genes encoding all but one of the Agl proteins are sequestered into a single gene island. The same region of the genome includes sequences also suspected but not yet verified as serving N-glycosylation roles, such as HVO_1526. In the following, HVO_1526, renamed AglS, is shown to be necessary for the addition of the final Mannose subunit of the pentasaccharide N-linked to the surface (S)-layer glycoprotein, a convenient reporter of N-glycosylation in Hfx. volcanii. Relying on bioinformatics, topological analysis, gene deletion, mass spectrometry, and biochemical assays, AglS was shown to act as a Dolichol Phosphate-Mannose mannosyltransferase, mediating the transfer of Mannose from Dolichol Phosphate to the tetrasaccharide corresponding to the first four subunits of the pentasaccharide N-linked to the S-layer glycoprotein.

  • glyco engineering in archaea differential n glycosylation of the s layer glycoprotein in a transformed haloferax volcanii strain
    Microbial Biotechnology, 2011
    Co-Authors: Doron Calo, Ziqiang Guan, Jerry Eichler
    Abstract:

    Summary Archaeal glycoproteins present a variety of N-linked glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific func- tions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Halof- erax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S- layer glycoprotein is modified by an N-linked pen- tasaccharide. In the following, S-layer glycoprotein N-glycosylationwasconsideredincellsinwhichAglD, the Dolichol Phosphate Mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacte- rium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer gly- coprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering 'workshop'.

  • Glyco-engineering in Archaea: differential N-glycosylation of the Slayer glycoprotein in a transformed Haloferax volcanii strain. Microb Biotechnol 4: 461–470. doi: 10. 1111/j.1751-7915.2011.00250.x PMID: 21338478
    2011
    Co-Authors: Doron Calo, Jerry Eichler, Ziqiang Guan, Department Of Life Sciences
    Abstract:

    glycans not seen elsewhere. The ability to harness the agents responsible for this unparalleled diversity offers the possibility of generating glycoproteins bearing tailored glycans, optimized for specific func-tions. With a well-defined N-glycosylation pathway and available genetic tools, the haloarchaeon Halof-erax volcanii represents a suitable platform for such glyco-engineering efforts. In Hfx. volcanii, the S-layer glycoprotein is modified by an N-linked pen-tasaccharide. In the following, S-layer glycoprotein N-glycosylation was considered in cells in which AglD, the Dolichol Phosphate Mannose synthase involved in addition of the final residue of the pentasaccharide, was replaced by a haloarchaeal homologue of AglJ, the enzyme involved in addition of the first residue of the N-linked pentasaccharide. In the engineering strain, the S-layer glycoprotein is modified by a novel N-linked glycan not found on this reporter from the parent strain. Moreover, deletion of AglD alone and introduction of the AglJ homologue from Halobacte-rium salinarum, OE2528R, into the deletion strain resulted in increased biosynthesis of the novel 894 Da glycan concomitant with reduced biogenesis of the pentasaccharide normally N-linked to the S-layer gly-coprotein. These findings justify efforts designed to transform Hfx. volcanii into a glyco-engineering ‘workshop’

Hisashi Ashida - One of the best experts on this subject based on the ideXlab platform.

  • DPM1, the Catalytic Subunit of Dolichol-Phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3
    The Journal of biological chemistry, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Taroh Kinoshita
    Abstract:

    Abstract Dolichol-Phosphate Mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-Mannose, and C-Mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.

  • mammalian pig x and yeast pbn1p are the essential components of glycosylphosphatidylinositol mannosyltransferase i
    Molecular Biology of the Cell, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Taroh Kinoshita
    Abstract:

    Within the endoplasmic reticulum (ER), Mannoses and glucoses, donated from Dolichol-Phosphate-Mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four Mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M. We found that Saccharomyces cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M. This is the first report of an essential subcomponent of glycosyltransferases using Dolichol-Phosphate-monosaccharide.

  • Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.
    Molecular biology of the cell, 2005
    Co-Authors: Hisashi Ashida, Yusuke Maeda, Yoshiko Murakami, Yeongjin Hong, Nobue Shishioh, Nakaba Sugimoto, Youn Uck Kim, Taroh Kinoshita
    Abstract:

    Within the endoplasmic reticulum (ER), Mannoses and glucoses, donated from Dolichol-Phosphate-Mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four Mannoses in the GPI-anchor precursors. We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M. The mutant gene, termed phosphatidylinositolglycan-class X (PIG-X), encoded a 252-amino acid ER-resident type I transmembrane protein with a large lumenal domain. PIG-X and PIG-M formed a complex, and PIG-M expression was